CN114181159A - Hydrazide derivative containing 2,4, 5-trisubstituted pyrimidine, preparation method and application thereof - Google Patents

Hydrazide derivative containing 2,4, 5-trisubstituted pyrimidine, preparation method and application thereof Download PDF

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CN114181159A
CN114181159A CN202111625343.3A CN202111625343A CN114181159A CN 114181159 A CN114181159 A CN 114181159A CN 202111625343 A CN202111625343 A CN 202111625343A CN 114181159 A CN114181159 A CN 114181159A
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amino
trisubstituted pyrimidine
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CN114181159B (en
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凌勇
冉凡胜
许中原
刘云
孟迟
邓学贤
张雪莹
姚俊辰
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Abstract

The invention belongs to the technical field of organic compound synthesis and medical application, and discloses a hydrazide derivative containing 2,4, 5-trisubstituted pyrimidine, and a preparation method and application thereof. The structure of the 2,4, 5-trisubstituted pyrimidine hydrazide derivative is shown as a general formula I
Figure DDA0003439514470000011
Wherein R is1Selected from C containing or not containing halogen, nitrogen, oxygen, or sulfur atoms1‑6Straight or branched chain alkyl, aryl; r2Selected from hydrogen, cyano, halogen, trifluoromethyl, methyl, methoxy. The compound has certain BTK/FLT3 dual inhibitory activity.

Description

Hydrazide derivative containing 2,4, 5-trisubstituted pyrimidine, preparation method and application thereof
Technical Field
The invention belongs to the technical field of organic compound synthesis and medical application, and relates to a hydrazide derivative containing 2,4, 5-trisubstituted pyrimidine, and a preparation method and application thereof.
Background
Bruton's Tyrosine Kinase (BTK) is a key node of the B Cell Receptor (BCR) and Fc receptor (FcR) signaling pathways. BCR and FcR are expressed on the surface of B cells and myeloid cells (macrophages, monocytes, mast cells, etc.), respectively. BTK regulates the survival and biological functions of B cells and myeloid cells through BCR and FcR signaling pathways, and is an important target for the treatment of various diseases involving B cell and/or macrophage abnormalities (see: Drug discovery today 2014; 19(8):1200 and Molcancer.2018; 17(1): 57). FMS-like tyrosine kinase 3(Fms-like tyrosine kinase 3, FLT3) belongs to type III receptor tyrosine kinase, and is mainly expressed in CD34+The surfaces of hematopoietic stem cells and immature hematopoietic progenitor cells play an important role in the development of hematopoietic stem cells and DC progenitor cells (see: physiologic reviews 2019; 99(3): 1433-. FLT3 is an important target for the treatment of hematologic malignancies and autoimmune diseases. Preclinical and clinical studies show that the double inhibition BTK and FLT3 have synergistic effect (see: Blood 2019; 134(Supplement _1):5477-5477), so that the design and synthesis of the novel BTK/FLT3 double-target drug with novel structure and drug property have important significance for treating hematological malignancy and autoimmune diseases.
Disclosure of Invention
The invention aims to provide 2,4, 5-trisubstituted pyrimidine hydrazide derivatives with BTK/FLT3 dual inhibitory activity, anti-inflammatory and anti-tumor effects, the compounds have excellent biological activity, and the molecular diversity and novelty of the compounds are improved; the invention also aims to provide a preparation method and application of the hydrazide derivative containing 2,4, 5-trisubstituted pyrimidine.
In order to achieve the purpose, the technical scheme disclosed by the invention is as follows:
in a first aspect of the present invention, the present invention provides a hydrazide derivative containing 2,4, 5-trisubstituted pyrimidine, or a pharmaceutically acceptable salt thereof, having the structure shown in formula I:
Figure BDA0003439514450000011
wherein R is1Selected from C containing or not containing halogen, nitrogen, oxygen, or sulfur atoms1-6Straight or branched chain alkyl, aryl; r2Selected from hydrogen, cyano, halogen, trifluoromethyl, methyl, methoxy; the substituted position of the hydrazide group is C-2 position, C-3 position and C-4 position.
Preferably, R1Selected from methoxyethyl, phenyl; r2Selected from fluorine;
preferably, the hydrazide derivative containing 2,4, 5-trisubstituted pyrimidine having the structure shown in the general formula I is selected from any one of the following compounds:
2- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) benzoyl hydrazine (I-1)
2- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) benzoyl hydrazine (I-2)
3- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) benzoyl hydrazine (I-3)
3- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) benzoyl hydrazine (I-4)
4- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) benzoyl hydrazine (I-5)
4- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) benzoyl hydrazine (I-6)
The parenthesis following the name of the above compound is the corresponding reference number, and for the convenience of description and the conciseness of expression, the above parenthesis will be directly applied to the following contents of the present specification.
The hydrazide derivative containing 2,4, 5-trisubstituted pyrimidine of the invention can exist in a free form or further in a salt form, and the purpose is to improve water solubility and increase bioavailability.
The pharmaceutically acceptable salt refers to conventional non-toxic salt, and mainly comprises salt formed by basic amino of 2,4, 5-trisubstituted pyrimidine hydrazide derivative. These salts are well known to those skilled in the art and the skilled artisan can prepare any pharmaceutically acceptable salt provided by the knowledge in the art. In addition, the skilled artisan may choose one salt and leave out another salt depending on solubility, stability, ease of formulation, etc. The determination and optimization of these salts is within the experience of the skilled artisan.
The invention also provides a method for preparing the hydrazide derivative containing 2,4, 5-trisubstituted pyrimidine shown in the general formula I, which comprises the following reaction scheme:
Figure BDA0003439514450000021
wherein R is1Selected from C containing or not containing halogen, nitrogen, oxygen, or sulfur atoms1-6Straight or branched chain alkyl, aryl; r2Selected from hydrogen, cyano, halogen, trifluoromethyl, methyl, methoxy; the substituted position of the hydrazide group is C-2 position, C-3 position and C-4 position.
Reagents and conditions: (a) methyl aminobenzoate, N, N-Diisopropylethylamine (DIPEA), isopropanol, 85 ℃ and 4 h; (b) substituted aniline, trifluoroacetic acid, n-butanol, 110 ℃,12 h; (c) hydrazine hydrate, methanol, 80 ℃,12 h.
The preparation method of the 2,4, 5-trisubstituted pyrimidine hydrazide derivative with the structure shown in the general formula I comprises the following steps:
(i) compound 1 and methyl aminobenzoate were dissolved in isopropanol, DIPEA was added, and reaction was carried out at 85 ℃ for 4 hours. TLC detection, complete reaction, cooling to room temperature, precipitation of a large amount of solid, filtration, and recrystallization of a filter cake with ethyl acetate to obtain an intermediate 2.
(ii) Dissolving the intermediate 2 in n-butanol, adding substituted aniline, dropwise adding trifluoroacetic acid into the solution, and reacting at 110 ℃ for 12 h. TLC detection, complete reaction, cooling to room temperature, reduced pressure evaporation to remove solvent, silica gel column chromatography to obtain intermediate 3.
(iii) The intermediate 3 is dissolved in methanol, hydrazine hydrate is added, and the reaction is carried out for 12h at 80 ℃. And (3) detecting by TLC (thin layer chromatography), cooling to room temperature, evaporating to remove the solvent under reduced pressure, and performing silica gel column chromatography to obtain a target compound I, namely the 2,4, 5-trisubstituted pyrimidine hydrazide-containing derivative with the structure shown in the general formula I.
The room temperature of the invention is 20-30 ℃.
The invention also provides a pharmaceutical composition which contains the 2,4, 5-trisubstituted pyrimidine-containing hydrazide derivative or a pharmaceutically acceptable salt thereof.
Pharmaceutical compositions of the compounds of the invention may be administered in any manner selected from: oral, aerosol inhalation, rectal, nasal, vaginal, topical, parenteral such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal or intracranial injection or infusion, or by means of an explanted reservoir, with oral, intramuscular, intraperitoneal or intravenous administration being preferred.
The invention also provides a pharmaceutical preparation which comprises the 2,4, 5-trisubstituted pyrimidine hydrazide derivative or pharmaceutically acceptable salt thereof or a composition containing the 2,4, 5-trisubstituted pyrimidine hydrazide derivative or pharmaceutically acceptable salt thereof and pharmaceutically acceptable auxiliary materials and/or carriers.
The hydrazide derivatives of 2,4, 5-trisubstituted pyrimidines of this invention or the pharmaceutical compositions containing them may be administered in unit dosage forms. The administration dosage form can be liquid dosage form or solid dosage form. The liquid dosage form can be true solution, colloid, microparticle, emulsion, or suspension. Other dosage forms such as tablet, capsule, dripping pill, aerosol, pill, powder, solution, suspension, emulsion, granule, suppository, lyophilized powder for injection, clathrate, landfill, patch, liniment, etc.
The pharmaceutical combination or pharmaceutical preparation of the present invention may further comprise a conventional carrier, wherein the pharmaceutically acceptable carrier includes but is not limited to: ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycerol, sorbates, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, beeswax, lanolin and the like. The carrier may be present in the pharmaceutical composition in an amount of 1% to 98% by weight, typically about 80% by weight. For convenience, the local anesthetic, preservative, buffer, etc. may be dissolved directly in the vehicle.
Oral tablets and capsules may contain excipients such as binding agents, for example syrup, acacia, sorbitol, tragacanth, or polyvinylpyrrolidone, fillers such as lactose, sucrose, corn starch, calcium phosphate, sorbitol, glycine, lubricants such as magnesium stearate, talc, polyethylene glycol, silica, disintegrants such as potato starch, or acceptable wetting agents such as sodium lauryl sulfate. The tablets may be coated by methods known in the art of pharmacy.
The oral liquid can be made into water and oil suspension, solution, emulsion, syrup, or dried product, and supplemented with water or other suitable medium before use. Such liquid preparations may contain conventional additives such as suspending agents, sorbitol, cellulose methyl ether, glucose syrup, gelatin, hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gelatin, hydrogenated edible fats and oils, emulsifying agents such as lecithin, sorbitan monooleate, gum arabic; or a non-aqueous carrier (which may comprise an edible oil), such as almond oil, an oil such as glycerol, ethylene glycol, or ethanol; preservatives, e.g. methyl or propyl p-hydroxybenzoates, sorbic acid. Flavoring or coloring agents may be added if desired.
Suppositories may contain conventional suppository bases such as cocoa butter or other glycerides.
For parenteral administration, liquid dosage forms are generally prepared from the compound and a sterile carrier. The carrier is preferably water. The compound can be dissolved in the carrier or made into suspension solution according to the concentration of the carrier and the drug, and the compound is firstly dissolved in water when made into the solution for injection, filtered and sterilized and then filled into a sealed bottle or ampoule.
It will be appreciated that the optimum dosage and interval for administration of the 2,4, 5-trisubstituted pyrimidine hydrazide-containing derivatives of formula I will be determined by the nature of the compound and external conditions such as the form, route and site of administration and the particular mammal being treated, and that such optimum dosage may be determined by conventional techniques. It will also be appreciated that the optimal course of treatment, i.e. the daily dosage of a compound of formula I over a nominal period of time, may be determined by methods well known in the art.
The invention also provides application of the 2,4, 5-trisubstituted pyrimidine hydrazide derivative or pharmaceutically acceptable salt thereof or a composition containing the 2,4, 5-trisubstituted pyrimidine hydrazide derivative or pharmaceutically acceptable salt thereof in preparation of BTK and/or FLT3 inhibitor drugs.
The invention also provides application of the 2,4, 5-trisubstituted pyrimidine hydrazide derivative or pharmaceutically acceptable salt thereof or a composition containing the 2,4, 5-trisubstituted pyrimidine hydrazide derivative or pharmaceutically acceptable salt thereof in preparing an anti-tumor medicament, wherein the tumor is lymphoma or leukemia.
The invention also provides application of the 2,4, 5-trisubstituted pyrimidine-containing hydrazide derivative or pharmaceutically acceptable salt thereof in preparing anti-inflammatory drugs. The hydrazide derivative containing 2,4, 5-trisubstituted pyrimidine is preferably 3- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) benzoyl hydrazine.
Compared with the prior art, the invention provides 2,4, 5-trisubstituted pyrimidine-containing hydrazide derivatives, a preparation method and application thereof, the compounds have novel structures, different from the prior art, the compounds show double inhibitory activity on BTK and FLT3, and representative compounds have strong double inhibitory activity IC on BTK and FLT350At low nanomolar level (IC)50<20 nM); the compound has antiproliferative activity on lymphoma and leukemia cell strains and is marketed as Ibrutinib and Sorafenib; in addition, representative compounds have certain anti-inflammatory effects. The compounds provided by the invention are useful inThe development of new antitumor and anti-inflammatory drugs.
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FIG. 1 shows the concentration of TNF-. alpha.A (A) and IL-6(B) in the supernatant of RAW264.7 cells.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. The following experimental examples are only for illustrating the technical effects of the present invention, but the experimental examples are not intended to limit the present invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
Example 1: preparation of intermediate 2
The starting materials 2, 4-dichloro-5-fluoropyrimidine (12mmol, 1.2eq), methyl aminobenzoate (10mmol, 1.0eq) and DIPEA (10mmol, 1.0eq) were dissolved in 20mL of isopropanol and reacted by heating at 85 ℃ for 4 hours. After the reaction is finished, the reaction liquid is cooled to room temperature, a large amount of solid is separated out, the solid is filtered, and a filter cake is recrystallized by ethyl acetate to obtain an intermediate 2.
Example 2: preparation of intermediate 3
Dissolving the intermediate 2(1mmol, 1eq) in 30mL of n-butanol, adding different substituted anilines (1.1mmol, 1.1eq), adding 5 drops of trifluoroacetic acid dropwise into the solution, and heating at 110 ℃ for reaction for 12 h. After the reaction, the reaction mixture was cooled to room temperature, the solvent was distilled off under reduced pressure, and silica gel column chromatography (dichloromethane/methanol 200:1 to 20:1) was performed to obtain intermediate 3.
3-1: methyl 2- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) benzoate
1H NMR(400MHz,DMSO-d6)δ10.93(d,J＝2.3Hz,1H),9.22(s,1H),8.91(d,J＝8.5Hz,1H),8.20(d,J＝3.2Hz,1H),8.03(dd,J＝8.0,1.6Hz,1H),7.63(dt,J＝8.0,1.6Hz,1H),7.53(d,J＝9.0Hz,2H),7.16(dt,J＝8.0,1.2Hz,1H),6.92–6.86(m,2H),4.09–4.03(m,2H),3.90(s,3H),3.68–3.63(m,2H),3.32(s,3H).13C NMR(101MHz,DMSO-d6)δ168.76,156.23,154.01,149.33(d,J＝11.2Hz),142.43,141.70,141.56(d,J＝17Hz),141.21(d,J＝244Hz),134.84,134.21,131.33,122.30,121.64,120.97,115.59,114.72,70.98,67.51,58.64,53.08.HRMS(ESI)m/z calcdfor C21H22FN4O4[M+H]+413.1620,found413.1621.
3-2: methyl 2- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) benzoate
1H NMR(400MHz,DMSO-d6)δ10.93(d,J＝2.2Hz,1H),9.43(s,1H),8.89(d,J＝8.5Hz,1H),8.24(d,J＝3.2Hz,1H),8.03(dd,J＝8.0,1.6Hz,1H),7.68(d,J＝9.0Hz,2H),7.65–7.58(m,1H),7.40–7.33(m,2H),7.21–7.13(m,1H),7.13–7.05(m,1H),7.03–6.94(m,4H),3.90(s,3H).13C NMR(101MHz,DMSO-d6)δ168.73,158.35,155.94(d,J＝2.7Hz),150.68,149.43(d,J＝9.7Hz),142.65,141.57,141.35,140.20,137.18,134.78,131.33,130.37,123.08,122.43,121.40,121.11,120.06,117.85,115.83,53.09.HRMS(ESI)m/z calcd for C24H20FN4O3[M+H]+431.1514,found431.1516.
3-3: 3- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) benzoic acid methyl ester
1H NMR(400MHz,DMSO-d6)δ9.54(s,1H),9.06(s,1H),8.21(t,J＝1.7Hz,1H),8.18(d,J＝8.3Hz,1H),8.11(d,J＝3.6Hz,1H),7.66(d,J＝7.8Hz,1H),7.54–7.42(m,3H),6.78(d,J＝9.0Hz,2H),4.02(dd,J＝5.4,3.8Hz,2H),3.83(s,3H),3.67–3.61(m,2H),3.31(s,3H).13C NMR(101MHz,DMSO-d6)δ166.65,156.18(d,J＝2.7Hz),153.62,149.90(d,J＝10.8Hz),141.61(d,J＝19.7Hz),140.74(d,J＝245Hz),139.85,134.41,130.41,129.39,126.25,123.97,122.39,121.09,114.54,70.96,67.41,58.62,52.59.HRMS(ESI)m/z calcd for C21H22FN4O4[M+H]+413.1620,found413.1620.
3-4: 3- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) benzoic acid methyl ester
1H NMR(400MHz,DMSO-d6)δ9.60(s,1H),9.29(s,1H),8.22(t,J＝1.8Hz,1H),8.18-8.14(t,J＝6.8Hz,2H),7.69–7.61(m,3H),7.47(t,J＝7.9Hz,1H),7.38–7.31(m,2H),7.08(t,J＝7.4Hz,1H),6.97–6.91(m,2H),6.88(d,J＝9.0Hz,2H),3.79(s,3H).13C NMR(101MHz,DMSO-d6)δ166.59,158.32,155.94(d,J＝2.9Hz),150.34,150.00(d,J＝10.9Hz),141.58(d,J＝19.6Hz),140.80(d,J＝245Hz),139.74,137.35,130.37(d,J＝6.9Hz),129.42,126.48,124.12,123.04,122.60,120.84,119.91,117.84,52.58.HRMS(ESI)m/z calcd for C24H20FN4O3[M+H]+431.1514,found431.1516.
3-5: 4- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) benzoic acid methyl ester
1H NMR(400MHz,DMSO-d6)δ9.67(s,1H),9.13(s,1H),8.15(d,J＝3.6Hz,1H),8.01(d,J＝8.8Hz,2H),7.88(d,J＝8.9Hz,2H),7.52(d,J＝9.0Hz,2H),6.87(d,J＝9.0Hz,2H),4.08–4.03(m,2H),3.84(s,3H),3.68–3.63(m,2H),3.32(s,3H).13C NMR(101MHz,DMSO-d6)δ166.39,155.99(d,J＝2.6Hz),154.07,149.66(d,J＝10.7Hz),144.22,141.29(d,J＝20.3Hz),140.82(d,J＝246Hz),134.06,130.30,123.63,121.88,120.26,114.72,70.94,67.49,58.62,52.36.HRMS(ESI)m/z calcd for C21H22FN4O4[M+H]+413.1620,found413.1621.
3-6: 4- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) benzoic acid methyl ester
1H NMR(400MHz,DMSO-d6)δ9.99(s,1H),9.59(s,1H),8.24(d,J＝4.0Hz,1H),7.98(d,J＝8.8Hz,2H),7.89(d,J＝8.8Hz,2H),7.61(d,J＝8.9Hz,2H),7.43–7.34(m,2H),7.11(t,J＝7.4Hz,1H),7.04–6.95(m,4H),3.83(s,3H).13C NMR(101MHz,DMSO-d6)δ166.25,157.88,154.14,152.12,150.84(d,J＝11.0Hz),143.24,140.48(d,J＝247Hz),137.37(d,J＝15.5Hz),135.43,130.46,130.25,124.69,123.43,123.14,121.19,119.85,118.22,52.45.HRMS(ESI)m/z calcd for C24H20FN4O3[M+H]+431.1514,found431.1516.
Example 3: preparation of the target Compound I
The ester-based compound I (1.0eq) was dissolved in 10mL of methanol, followed by addition of hydrazine hydrate (10eq) and reaction at 80 ℃ for 12 hours. After the reaction is finished, the reaction product is cooled to room temperature, the solvent is removed by evaporation under reduced pressure, and silica gel column chromatography (dichloromethane/methanol is 100:1-10:1) is carried out to obtain the target compound I.
I-1: 2- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) benzoyl hydrazine
1H NMR(400MHz,DMSO-d6)δ11.56(s,1H),10.12(s,1H),9.16(s,1H),8.80(d,J＝8.3Hz,1H),8.14(d,J＝3.3Hz,1H),7.73(dd,J＝7.9,1.1Hz,1H),7.54(d,J＝9.0Hz,2H),7.49(t,J＝8.5Hz,1H),7.10(t,J＝7.1Hz,1H),6.89(d,J＝8.8Hz,2H),4.66(s,2H),4.09–4.02(m,2H),3.69–3.62(m,2H),3.31(s,3H).13C NMR(101MHz,DMSO-d6)δ168.13,156.24(d,J＝3.1Hz),153.89,149.35(d,J＝10.0Hz),141.22(d,J＝244Hz),142.44,141.12(d,J＝18.7Hz),139.92,134.35,132.19,128.14,122.11,121.48,121.06,119.24,114.69,70.98,67.50,58.64.HRMS(ESI)m/z calcd for C20H22FN6O3[M+H]+413.1732,found 413.1722.
I-2: 2- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) benzoyl hydrazine
1H NMR(400MHz,DMSO-d6)δ11.59(s,1H),10.12(s,1H),9.37(s,1H),8.81(d,J＝8.4Hz,1H),8.18(d,J＝3.2Hz,1H),7.71(dd,J＝19.0,8.4Hz,3H),7.52–7.43(m,1H),7.41–7.31(m,2H),7.13–7.05(m,2H),7.02–6.94(m,4H),4.64(s,2H).13C NMR(101MHz,DMSO-d6)δ168.10,158.38,155.99(d,J＝3.0Hz),150.57,149.44(d,J＝10.2Hz),141.45(d,J＝245Hz),141.03(d,J＝18.7Hz),139.83,137.32,132.15,130.36,128.16,123.06,122.24,121.30,121.14,120.08,119.41,117.82.HRMS(ESI)m/z calcd for C23H20FN6O2[M+H]+431.1626,found431.1620.
I-3: 3- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) benzoyl hydrazine
1H NMR(400MHz,DMSO-d6)δ9.74(s,1H),9.45(s,1H),9.04(s,1H),8.08(d,J＝3.7Hz,1H),8.03(t,J＝1.7Hz,1H),7.95(d,J＝8.1Hz,1H),7.49(d,J＝9.0Hz,3H),7.39(t,J＝7.9Hz,1H),6.78(d,J＝9.1Hz,2H),4.49(s,2H),4.10–3.98(m,2H),3.69–3.60(m,2H),3.31(s,3H).13C NMR(101MHz,DMSO-d6)δ166.45,156.22(d,J＝2.7Hz),153.58,150.08(d,J＝10.8Hz),142.00,141.39(d,J＝19.1Hz),139.49(d,J＝13.2Hz),134.43(d,J＝5.6Hz),128.88,124.53,121.89,121.09(d,J＝17.2Hz),114.59,70.97,67.41,58.63.HRMS(ESI)m/z calcd for C20H22FN6O3[M+H]+413.1732,found 413.1724.
I-4: 3- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) benzoyl hydrazine
1H NMR(400MHz,DMSO-d6)δ9.73(s,1H),9.51(s,1H),9.27(s,1H),8.12(d,J＝3.6Hz,1H),8.03(t,J＝1.7Hz,1H),7.97(d,J＝8.0Hz,1H),7.65(d,J＝9.0Hz,2H),7.49(d,J＝7.8Hz,1H),7.43–7.30(m,3H),7.07(t,J＝7.4Hz,1H),6.99–6.83(m,4H),4.45(s,2H).13C NMR(101MHz,DMSO-d6)δ166.38,158.42,155.98(d,J＝2.8Hz),150.18(t,J＝5.4Hz),142.21,141.34(d,J＝19.4Hz),139.76,139.36,137.46,134.39,130.33,128.89,124.72,122.96,121.98,121.26,120.78,120.08,117.72.HRMS(ESI)m/z calcd for C23H20FN6O2[M+H]+431.1626,found 431.1620.
I-5: 4- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) benzoyl hydrazine
1H NMR(400MHz,DMSO-d6)δ9.70(s,1H),9.52(s,1H),9.12(s,1H),8.12(d,J＝3.7Hz,1H),7.92(d,J＝8.7Hz,2H),7.79(d,J＝8.8Hz,2H),7.53(d,J＝9.0Hz,2H),6.87(d,J＝9.1Hz,2H),4.47(s,2H),4.10–4.02(m,2H),3.68–3.63(m,2H),3.31(s,3H).13C NMR(101MHz,DMSO-d6)δ166.07,156.23(d,J＝2.8Hz),153.80,149.70(d,J＝10.6Hz),142.28,141.54(d,J＝18.8Hz),140.86(d,J＝246Hz),134.42,127.92,127.55,121.30,120.22,114.71,70.98,67.50,58.64.HRMS(ESI)m/z calcd for C20H22FN6O3[M+H]+413.1732,found 413.1724.
I-6: 4- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) benzoyl hydrazine
1H NMR(400MHz,DMSO-d6)δ9.69(s,1H),9.56(s,1H),9.33(s,1H),8.16(d,J＝3.6Hz,1H),7.92(d,J＝8.8Hz,2H),7.79(d,J＝8.8Hz,2H),7.68(d,J＝9.0Hz,2H),7.41–7.32(m,2H),7.08(t,J＝7.4Hz,1H),7.04–6.89(m,4H),4.46(s,2H).13C NMR(101MHz,DMSO-d6)δ166.06,158.38,155.98(d,J＝2.8Hz),150.41,149.77(d,J＝10.8Hz),142.19,141.47(d,J＝19.7Hz),141.06(d,J＝246Hz),137.39,130.38,127.92,127.69,123.01,121.13,120.35,120.11,117.73.HRMS(ESI)m/z calcd for C23H20FN6O2[M+H]+431.1626,found 431.1620.
Experimental example: test for BTK and FLT3 inhibitory activity, anti-proliferation activity of tumor cells and experiment for inhibiting proinflammatory cytokine release from inflammatory cells
1) Compound activity assay for BTK, FLT3 kinase inhibition:
experimental materials and instruments: this experiment was performed with the aid of Eurofins Pharma, british.
The experimental method comprises the following steps: all compounds tested were formulated in DMSO as 50-fold final assay concentration working solution. Compound working solution was first added as a first component to the test wells, followed by addition of kinase buffer diluted BTK or FLT3 kinase solution. The addition of Mg/ATP initiates the kinase reaction. Subsequently, the reaction was incubated at room temperature for 40 minutes, and a 0.5% phosphoric acid solution was added to terminate the reaction. 10 μ L of the reaction was spotted onto a pad of P30 filter paper, washed 4 times with 0.425% phosphoric acid for 4 minutes each, then washed once with methanol, followed by drying and scintillation counting.
The test was set up with a compound test group (C), a positive control group (P) and a blank control group (B). The positive control group contained no test compound, DMSO was used instead (final concentration 2%), and the other components were identical to the test group (residual kinase activity 100%); staurosporine (staurosporine) was used in place of test compound in the blank control group to eliminate kinase activity and establish a baseline (residual kinase activity 0%).
Figure BDA0003439514450000061
IC was calculated by fitting a curve using Gragopd prism6.0 software with the logarithm of concentration as the abscissa and the inhibition ratio as the center50The value is obtained. The test results of the target compound on the BTK and FLT3 kinase inhibition activity are shown in the table 1.
TABLE 1 inhibitory Activity of the Compounds of interest on BTK and FLT3 kinase
Figure BDA0003439514450000062
A: the inhibition rate is more than 60%; b, 60% > inhibition rate is more than 40%; c, the inhibition rate is less than 40 percent
Table 1 shows that most compounds have stronger inhibitory activity to BTK and FLT3 (the inhibition rate of kinase at 1 mu M concentration)>60%). The compounds I-1, I-3, I-4, I-5 and I-6 have strong double inhibition effects on BTK and FLT3, and representative compounds have strong double inhibition activities on BTK and FLT3, namely IC50At low nanomolar level (IC)50<20nM)。
2) Growth inhibitory activity of compounds on tumor cells:
experimental materials and instruments: jeko-1 and RAW264.7 cell strains, RPMI-1640 culture medium, fetal bovine serum, PBS buffer solution, penicillin sodium (10000units/mL) -streptomycin sulfate (10mg/mL), a CCK-8 kit, an inverted optical microscope, a cell culture box, a super clean bench, a bench centrifuge, a microplate reader and an ultra-low temperature refrigerator.
The experimental method comprises the following steps:
inoculating the tumor cells in logarithmic growth phase in 96-well culture plate with the number of cells being 1 × 104Perwell, measured by addition of different concentrationsThe cell culture solution of the compound is simultaneously provided with a positive control group and a DMSO blank control group, and the concentration of DMSO is adjusted to be less than or equal to 1 per mill. Each concentration is provided with 3 compound holes, after the addition is finished, the mixture is placed at 37 ℃ and 5 percent CO2Incubate in the incubator for 72 h. Then 20. mu.L of CCK-8 solution was added to each well and the plates were placed at 37 ℃ in 5% CO2Continuously incubating for 1-4h in a constant temperature incubator, measuring absorbance value of the sample at 450nm wavelength by using an enzyme-labeling instrument, normalizing the obtained value and a negative DMSO control group, and calculating IC by using Prism6.0 software50The value is obtained.
Cell survival% (% OD administration-OD blank)/(OD positive control-OD blank) × 100%.
TABLE 2 inhibition of Jeko-1 and RAW264.7 cell growth by compounds of interest
Figure BDA0003439514450000063
Figure BDA0003439514450000071
IC50: half maximal inhibitory concentration
A：IC50<2μM；B:2μM<IC50<20μM；C:20μM<IC50
Table 2 experimental data show that most compounds have significant antiproliferative activity, IC, on Jeko-1 cells50The values were comparable to or even lower than the positive control drug, imatinib, and most compounds had moderate-intensity antiproliferative activity on RAW264.7 cells. Half-inhibitory concentrations of compound I-3 were at low micromolar (less than 2. mu.M) on both cell lines.
3) Experiment of inhibiting inflammatory cell from secreting inflammatory cytokine by compound:
experimental materials and instruments: RAW264.7 cell strain, RPMI-1640 culture medium, fetal bovine serum, PBS buffer solution, penicillin sodium (10000units/mL) -streptomycin sulfate (10mg/mL), an inverted optical microscope, a cell culture box, an ultra-clean workbench, a desktop centrifuge, a microplate reader, an ultra-low temperature refrigerator and an Elisa kit.
The experimental method comprises the following steps:
immune cells in logarithmic growth phase (RAW264.7) were seeded in 96-well plates and cultured for 12 hours. Old medium was discarded and new medium containing LPS (final concentration of LPS 1. mu.g/mL) was added to each well. The experiment was divided into a blank control group, an LPS group, a compound group, and an Ibrutinib group. And (3) discarding the old culture medium after culturing for 24 hours, adding fresh culture media containing test drugs with various concentrations according to experimental groups, continuously culturing for 24 hours, collecting the supernatant, centrifuging at the rotating speed of 1000rpm for 10 minutes at 4 ℃ to remove cell precipitates, carefully sucking the cell supernatant into a clean EP tube, subpackaging, storing in a refrigerator at-80 ℃ and detecting. Cytokine levels such as ILs, IFNs, TNF-alpha in cell supernatants were measured using an Elisa commercial kit. The results of the detection are shown in FIG. 1.
As shown in fig. 1: LPS induces RAW264.7 cells to secrete TNF-alpha and IL-6 cytokine levels to be obviously increased; the compound I-3 can inhibit inflammatory cells from secreting TNF-alpha and IL-6 in a dose-dependent mode, the capacity of inhibiting inflammatory cell factor secretion is equivalent to that of positive drug Imatinib (IBN), and the I-3 has a potential anti-inflammatory effect.
The above description is only a preferred embodiment of the present disclosure and is not intended to limit the present disclosure, and various modifications and changes may be made to the present disclosure by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present disclosure should be included in the protection scope of the present disclosure.

Claims (10)

1. The 2,4, 5-trisubstituted pyrimidine-containing hydrazide derivative has a structure shown as a general formula I:
Figure FDA0003439514440000011
wherein R is1Selected from C containing or not containing halogen, nitrogen, oxygen, or sulfur atoms1-6Straight or branched chain alkyl, aryl; r2One selected from hydrogen, cyano, halogen, trifluoromethyl, methyl and methoxy; the substituted position of the hydrazide group is C-2 position, C-3 or C-4.
2. The hydrazide derivative containing 2,4, 5-trisubstituted pyrimidine as claimed in claim 1, wherein R is1Selected from methoxyethyl or phenyl; r2Selected from fluorine.
3. The hydrazide derivative containing 2,4, 5-trisubstituted pyrimidine according to claim 2, selected from the group consisting of:
2- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) benzoyl hydrazine;
2- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) benzoyl hydrazine;
3- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) benzoyl hydrazine;
3- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) benzoyl hydrazine;
4- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) benzoyl hydrazine;
4- ((5-fluoro-2- ((4-phenoxyphenyl) amino) pyrimidin-4-yl) amino) benzoyl hydrazine.
4. A preparation method of 2,4, 5-trisubstituted pyrimidine hydrazide derivatives is characterized by taking a compound 1 and aminobenzoate as starting materials to prepare a compound with a structure shown in a general formula I through the following reaction route:
Figure FDA0003439514440000012
wherein R is1Selected from C containing or not containing halogen, nitrogen, oxygen, or sulfur atoms1-6Straight or branched chain alkyl, aryl; r2Selected from hydrogen, cyano, halogen, trifluoromethyl, methyl, methoxy; the substituted position of the hydrazide group is C-2, C-3 or C-4;
the preparation method comprises the following steps:
s1, dissolving the compound 1 and methyl aminobenzoate in isopropanol, adding DIPEA, and reacting for 4 hours at 85 ℃. TLC detection, complete reaction, cooling to room temperature, separating out a large amount of solid, filtering, and recrystallizing a filter cake with ethyl acetate to obtain an intermediate 2;
s2, dissolving the intermediate 2 in n-butanol, adding substituted aniline, dropwise adding trifluoroacetic acid into the solution, and reacting at 110 ℃ for 12 hours. TLC detection, complete reaction, cooling to room temperature, reduced pressure evaporation to remove the solvent, silica gel column chromatography to obtain an intermediate 3;
s3, dissolving the ester compound I in methanol, adding hydrazine hydrate, and reacting at 80 ℃ for 12 h. And (3) detecting by TLC (thin layer chromatography), cooling to room temperature, evaporating to remove the solvent under reduced pressure, and performing silica gel column chromatography to obtain a target compound I, namely the 2,4, 5-trisubstituted pyrimidine hydrazide-containing derivative.
5. A pharmaceutical composition comprising the hydrazide derivative containing 2,4, 5-trisubstituted pyrimidine according to any one of claims 1 to 3 and/or a pharmaceutically acceptable salt thereof.
6. A pharmaceutical preparation, which comprises an active ingredient and pharmaceutically acceptable excipients and/or carriers, wherein the active ingredient comprises the hydrazide derivative containing 2,4, 5-trisubstituted pyrimidine as claimed in any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.
7. Use of a 2,4, 5-trisubstituted pyrimidine-containing hydrazide derivative according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof in the preparation of a BTK and/or FLT3 inhibitor drug.
8. The application of the 2,4, 5-trisubstituted pyrimidine-containing hydrazide derivative or pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 3 in the preparation of anti-tumor drugs, wherein the tumor is lymphoma or leukemia.
9. Use of the 2,4, 5-trisubstituted pyrimidine-containing hydrazide derivative according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof in the preparation of an anti-inflammatory agent.
10. The use of claim 9, wherein the 2,4, 5-trisubstituted pyrimidine-containing hydrazide derivative is 3- ((5-fluoro-2- ((4- (2-methoxyethoxy) phenyl) amino) pyrimidin-4-yl) amino) benzoyl hydrazine.
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